Although the level of prostaglandin (PG) D(2) in cerebrospinal fluid (CSF) affects the action of D-type prostanoid receptors that promote physiological sleep, the regulatory system of PGD(2) clearance from the CSF is not fully understood. The purpose of this study was to investigate PGD(2) elimination from the CSF via the blood-CSF barrier (BCSFB). The in vivo PGD(2) elimination clearance from the CSF was 16-fold greater than that of inulin, which is considered to reflect CSF bulk flow. This process was inhibited by the simultaneous injection of unlabeled PGD(2). The characteristics of PGD(2) uptake by isolated choroid plexus were, at least partially, consistent with those of PG transporter (PGT) and organic anion transporter 3 (OAT3). Studies using an oocyte expression system showed that PGT and OAT3 were able to mediate PGD(2) transport with a Michaelis-Menten constant of 1.07 and 7.32 μM, respectively. Reverse transcription-polymerase chain reaction and immunohistochemical analyses revealed that PGT was localized on the brush-border membrane of the choroid plexus epithelial cells. These findings indicate that the system regulating the PGD(2) level in the CSF involves PGT- and OAT3-mediated PGD(2) uptake by the choroid plexus epithelial cells, acting as a pathway for PGD(2) clearance from the CSF via the BCSFB.